Method for adjusting a gradient delay volume

ABSTRACT

The invention relates to a method for setting a gradient delay volume GDV of a liquid chromatography system for a chromatography run in liquid chromatography, in particular a high-performance liquid chromatography system, in which a desired value GDV target  of a gradient delay volume of the liquid chromatography system is ascertained or predefined and, if the value GDV target  deviates from a specific fixed value GDV actual  of a liquid chromatography system, this value GDV target  is set in a range 0≤ΔGDV=GDV target −GDV actual ≤V max  of a volume of a volume adjustment device  5 . Furthermore, the invention relates to an automatic sampler for carrying out such a method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 and claims thepriority benefit of co-pending U.S. patent application Ser. No.14/661,793, filed Mar. 18, 2015, which claims the priority benefit under35 U.S.C. § 119 to German Patent Application No. 10 2014 103 766.9, byHermann Hochgraeber and Thomas Wachinger for “A method of adjusting agradient delay volume” filed on Mar. 19, 2014, the disclosure of whichis incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of liquid chromatography, inparticular high-performance liquid chromatography (HPLC).

BACKGROUND

Liquid chromatography (in particular HPLC) is used for the purpose ofseparating liquid samples into their components by means of achromatography column. In this case, the separating power of the(separating) column is dependent, inter alio, on its length and on theparticle size of the packing material. For the best possible separation,columns having a sufficient length and a small particle size arerequired. Such columns have a high flow resistance and therefore requiresubstantially higher pressures for operation than conventional columns.

Furthermore, a sufficiently rapid separation is desirable, to enable ahigh sample throughput. This requires a high flow speed in the column,whereby the counterpressure in the column also increases.

One possibility for accelerating the separation or increasing theseparating power is to change the solvent composition over the durationof the separation (referred to hereafter as the gradient, i.e., thedegree of the change of the elution force or the proportion of a solventwith time is referred to as the gradient or solvent gradient).

Two different technical implementations for achieving a gradient haveprevailed due to the different requirements for the separation. Theseare so-called high-pressure gradient forming (HPG) and low-pressuregradient forming (LPG). High-pressure gradient forming operates by meansof two independent pumps, which are connected to one another via aT-part on the high-pressure side of the system. The gradient isgenerated by way of the change of the flow rates at the two pumps.Low-pressure gradient forming only requires one pump havingproportioning valve unit connected upstream. During the aspirating cycleof the pump, the various solvents are drawn successively into the pumpby opening and closing the valves (for example, solenoid valves) in theproportioning valve unit. The gradient is formed due to the variationsin the opening times for the various solvents. To smooth out compositionfluctuations in the solvent mixture (mixture irregularity), a mixer isconnected downstream from the pump as a standard feature.

In the various designs, there are different so-called gradient delayvolumes (GDV—or also dwell volume). The gradient delay volume or mixingvolume is dimensioned, on the one hand, by way of the holding capacityof all interconnected components from the mixing point up to the entryof the column. In the case of LPG, the GDV—depending on theimplementation of the switching valve and the connecting ports which areswitched through—is formed, for example, by the volumes of the followingcomponents (or a part thereof): pump head, mixer, connectingcapillaries, sample loop, switching valve, metering device. In the caseof HPG, the GDV—depending on the implementation of the switching valveand the connecting ports which are switched through—is formed, forexample, by the volumes of the following components (or a part thereof):T-part, mixer, connecting capillaries, sample loop, switching valve,metering device. On the other hand, the washing-out volume must also beconsidered, which results due to the flow properties of the components.

If a chromatographic method was developed on a specific HPLC system, itusually cannot be transferred to another system without problems. Thereproduction of a published method is also just as difficult. One causeof this is the different GDVs, which are accompanied by a shift and/orspreading of the retention times.

Furthermore, the mixture irregularity is also dependent on the GDV. Thehigher the GDV, the better the various solvents are mixed and the lessthe mixture irregularity.

The requirements for the GDV and the remaining mixture irregularity arealso strongly dependent on the application and the detector type used.Non-optical detectors, such as MS, ELSD, or CAD, are typicallyinsensitive to mixture irregularities, since these do not generatesignal variations due to differing detection sensitivities to theindividual solvent components. A mixer having small GDV with moderatemixer performance would therefore be acceptable in such a case. Thesituation appears very different, however, upon the use of a UVdetector, above all at low wavelengths. Extremely small variations ofthe solvent composition have effects here in visible variations in thebaseline. This makes it more difficult to determine the materialconcentration from the detector signal. A mixer having the highestpossible mixing efficiency with correspondingly greater GDV would beadvantageous in this case.

An unnecessarily large GDV is also advantageous, since the analysis,washing, and equilibration phases would also be increased unnecessarily.

If a method is transferred to another HPLC system, which has deviatingGDVs, an offset time (tgdv=Vgdv/flow rate) could be calculated and thegradient forming could be started delayed or early accordingly.

However, problems with the irregularity cannot be completely remedied inthis way. For this purpose, the mixer is normally manually replaced withanother mixer. Of course, automation of such a replacement by means of aswitching valve would also be conceivable. This solution would becumbersome because of the size of the mixers and additionally costly,however.

SUMMARY

The present invention is therefore based on the object of providing amethod and a system or an automatic sampler for carrying out thismethod, which enable an adaptation of the GDV or mixing volume in acost-effective and structurally simple manner.

According to the invention, in a chromatography system between mixingpoint (gradient occurrence) and entry of a separating column, a volumeadjustment device is provided, which enables simple, automated, andcost-effective change of the GDV or mixing volume. Such a volumeadjustment device offers a preferably continuously settable volume, forexample, via a control device, through which a solvent (gradient), whichchanges in composition, flows through during a chromatography run andcorrespondingly counts for the GDV.

Depending on the implementation of the chromatography system, componentsare located in the path—in or through which a gradient is navigated—forexample:

in LPG—depending on the implementation of the switching valve and theconnecting ports which are switched through: pump head, mixer,connecting capillaries, sample loop, switching valve, metering device,or parts thereof and;

in HPG—depending on the implementation of the switching valve and theconnecting ports which are switched through: T-part, mixer, connectingcapillaries, sample loop, switching valve, metering device, or partsthereof.

The holding capacity or the internal volume through which a gradientflows of the components located in each case in this path thereforeresult in a specific GDV for a respective system.

By way of the method according to the invention and by way of the liquidchromatography system according to the invention it is now possible toprovide a desired GDV—deviating from a system-specific (fixed) GDV.Thus, a predefined GDV, which is ascertained during a method developmentor is known, for example, of another system can be set, withoutcomponents (mixers, etc.) having to be changed, added, replaced, orswitched for this purpose on the present system. The change of the GDVis performed in this case in a range between the maximum and minimumadjustable volumes, for example, 0 μL to 1000 μL, in particular 0 μL to500 μL, preferably 0 μL to 120 μL, of the volume adjustment device,preferably by means of an electronic control unit.

In a further embodiment of the invention, the desired value iscontinuously adjustable by corresponding adjustment of the volumeadjustment device. Of course, it is also conceivable to provide discretesteps within the variable range of the GDV, however.

In a preferred embodiment of the invention, a metering device which isalready provided in the system, in particular in the automatic sampler,for taking samples and/or injecting samples, is used as the volumeadjustment device. The additional use of an already provided componentenables a structural and cost-reducing savings potential.

In an arbitrary embodiment of the invention, the desired GDV can bepreset before a chromatography run (in which a gradient is navigated),for example, before taking a sample or before injecting a sample, or canbe set during an injection or during a chromatography run.

In an arrangement (or with an automatic sampler), in which the meteringdevice or the holding capacity thereof counts for the GDV, in this way,the GDV can be changed by a preset holding capacity (greater than 0 ordeviating from a system-specific starting setting for the removal)before taking the sample, since this preset volume, which deviates fromthe standard position, counts for the GDV after switching over the paths(removal—injection).

Of course, however, it is also conceivable to set or vary the GDV duringand/or after a sample injection. Preferably, a pump device (also alreadyprovided in a chromatography system), in particular a solvent pump, canbe activated in opposition accordingly in this case, so that theresulting volume stream or the flow rate and/or the pressure in the pathto the separating column, in particular before the entry thereof, iskept essentially constant (i.e., with a deviation from the existing flowrate or the existing pressure of less than 20%, for example, less than10%, in particular less than 5%, preferably less than 1%) and damage tothe separating column can be avoided and the reproducibility of theanalysis can be ensured.

If the change of the GDV is performed, viewed in the flow direction tothe column, after an introduced sample or a so-called sample plug (i.e.,in the region between mixing point or occurrence of the gradient andinjection point), the run time of the gradient also changes in relationto the position of the sample plug. Correspondingly, during theanalysis, for example, during the increase of the GDV, chronologicalspreading or stretching of the result occurs (or of the components ofthe sample detected as peaks over time). However, if the change of theGDV or ΔGDV takes place, viewed in the flow direction of the column,before an introduced sample or a so-called sample plug (i.e., in theregion between the injection point and entry of the separating column),the run time of the gradient remains unchanged in relation to the sampleplug (no spreading), however, the result (or the detected peaks overtime) occurs with a delay with the factor ΔGDV/flow rate.

If an already provided metering device is used as the volume adjustmentdevice and the gradient flows through the metering device, a volumewhich is additionally set or varied in addition to a predefined samplevolume counts for the GDV. In this embodiment of the invention, it ispossible to set the desired GDV in a range from 0 or minimum of themetering device up to its maximum minus the predefined sample volume.

In a further embodiment of the invention, the smallest volume of thevolume adjustment device can be set at the end of a chromatography runaccording to this, to shorten the time for washing out to a minimum.

In addition to the above-mentioned advantages (setting the GDVcontinuously and without manual intervention, cost-effective usage of anexisting component for this purpose, possible saving of a mixer orassistance of the mixer), an undesired, excessively large GDV in thesystem can also be avoided or remedied according to the invention, bysimply changing the GDV or the mixing volume. In any case, it ispossible according to the invention, in contrast to conventional methodsand systems, to meet all application-specific requirements for GDV andmixing performance, without replacing the corresponding components.

Although the GDV or mixing volume during a chromatography run isconstant in conventional methods, it is furthermore also conceivableaccording to the invention to vary the GDV over the time of theanalysis. If one block in HPG, for example, requires very little solventin contrast to the other block, a higher GDV is then required tosuppress the mixture irregularity. At another point in time, when bothblocks require approximately equal amounts, a lesser GDV can besufficient.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be explained in greater detail hereafter on the basisof an exemplary embodiment illustrated in the drawing.

FIG. 1 shows two diagrams to illustrate the effect of two different GDVsduring a chromatography run on the detected result.

FIG. 2 shows a schematic illustration of an HPLC system having anautomatic sampler according to the invention, to which a chromatographycolumn is connected, wherein the injection valve is located in theINJECT position and an equilibration phase takes place in theillustrated state.

FIG. 3 shows the HPLC system in FIG. 2, wherein the injection valve wasswitched from the INJECT position into the PRESSURE EQUALIZATIONposition and the sample loop was switched out of the analytical path.

FIG. 4 shows the HPLC system in FIG. 3, wherein the injection valve wasswitched into the WASTE position.

FIG. 5 shows the HPLC system in FIG. 4, wherein the sample needle wasmoved into the sample container to take a sample and subsequently theplunger of the syringe was moved into the end position (position C) toaspirate the sample volume.

FIG. 6 shows the HPLC system according to FIG. 5, wherein the injectionvalve was switched from the LOAD position into the PRESSURE EQUALIZATIONposition and subsequently the plunger was moved into the position B forpressure equalization (pressure increase) in the sample loop.

FIG. 7 shows the HPLC system in FIG. 6, wherein the injection valve wasswitched from the PRESSURE EQUALIZATION position into the INJECTposition.

DETAILED DESCRIPTION OF EMBODIMENTS

The diagrams illustrated in FIG. 1 show how a change of the GDV effectsan analysis over time during a (sample) injection at the point in time 0minutes. A solvent composition over time is shown as a dashed line inthe diagrams. The solvent composition changes (transitions in the formof inflection points) from a constant (lower or front horizontalsection) lower limit (acetonitrile 40%) via a varying (from 40% to 70%acetonitrile) composition or gradient (linear, rising, middle region) upto a constant (horizontal upper or rear section) upper limit(acetonitrile 70%). As the gradient, for example, a gradient having anincrease of a solvent proportion B, for example, acetonitrile ACN, from40%-70% in 16 seconds (and corresponding reduction of the solventproportion A or the remaining solvent proportions from 60%-30%) is used.In the upper diagram (case A), the preset pump volume of the volumeadjustment device is V_(A)=25 μL, i.e., the GDV is increased by 25 μL,while in contrast in the lower diagram (case B), the adjusted pumpvolume is V_(A)=100 μL, i.e., the GDV is increased by 100 μL.

Since the increase of the GDV (ΔGDV=75 μL=100 μL−25 μL) from case A tocase B by the volume adjustment device, in particular a metering device5 in the exemplary embodiment, viewed in the flow direction toward the(separating) column 41, with respect to the position of an introducedsample plug, is performed after the sample plug (i.e., following therear separating surface between sample plug and solvent or betweenmixing point and tip of the sample needle 42), the incidence andtherefore effect of the gradient or of a respective concentration withrespect to the sample is shifted to the entry of the separating column41, without the point in time of the incidence of the sample itselfbeing delayed. As a result of the time shift or delay of the incidenceof the respective concentration at the entry of the column 41, however,the eluting effect of the gradient on components to be eluted of thesample is shifted or delayed. As a result thereof, chronologicalspreading or stretching of the (sample) components detected as peaksoccurs, which increases with components which are more difficult toelute (in the direction of the time axis).

As is apparent in FIG. 1 (lower diagram) as a detail of an analysisresult (with illustrated peak 2 to peak 7 in mAU=milli absorption unit),the individual peaks occur with greater delay with increasing time inrelation to their occurrence in the upper diagram, the later thecorresponding components are eluted (due to a higher concentration). Thepoints in time of the occurrence of the peaks increase accordingly asshown hereafter.

Peak 2 from 26.090 s to 26.450 s delay Δt = 0.360 s Peak 3 from 45.150 sto 47.250 s delay Δt = 2.100 s Peak 4 from 58.520 s to 62.780 s delay Δt= 4.260 s Peak 5 from 68.390 s to 73.630 s delay Δt = 5.240 s Peak 6from 71.960 s to 77.560 s delay Δt = 5.600 s Peak 7 from 79.430 s to85.520 s delay Δt = 6.090 s

The minimal delay at peak 2 is less than a measurement tolerance in thiscase, so that the occurrence thereof in case A to case B can beconsidered to be simultaneous or without delay. The reason for this isthat the isocratic part of the solvent was already eluted at this peak2, without a gradient already acting at this point in time.

In contrast, the amplitude or the maximum of the absorption can remainthe same or be reduced, as is apparent from FIG. 1.

As a result of the GDV change ΔGDV, the transitions or inflection pointsshift to the right between horizontal (isocratic) and the linear slope(gradient) on the time axis in the case of the transition horizontal toslope from 31 seconds to 36 seconds and in the case of the transitionslope to horizontal from 47 seconds to 52 seconds in the comparison ofthe upper diagram to the lower diagram of FIG. 1. This delay ofapproximately 5 seconds results due to the higher GDV (lower diagram) byΔGDV=75 μL and the flow rate of 0.95 mL/min (=15.83 μL/s).

In contrast, if the increase of the GDV, viewed in the flow directiontoward the column 41, takes place in position before the sample plug(i.e., between needle seat or injection port 45 and entry of theseparating column 41 or before the front separating surface betweensample plug and solvent), the detected result or peak of the componentsof the sample is delayed in time or shifted to the right in the diagramby the changed runtime without stretching.

FIG. 2 to FIG. 7 show, in a schematic illustration, an HPLC systemhaving an automatic sampler 10 operating according to the split loopprinciple, which has a metering device 5, an injection valve 3, and ahigh-pressure pump 40. In addition, the automatic sampler 10 has asample loop, which consists of a first connecting part 51, a secondconnecting part 52, 44, and a pump volume V (depending on the plungerposition, minimum, V_(A), V_(B), or V_(C) up to maximum V_(max), of, forexample, 120 μL). This can be a pressure-resistant line having a smalldiameter, for example, in the form of glass capillaries or stainlesssteel capillaries. The connecting part 51 is connected to a first sampleloop port 16 of the injection valve 3 and to the sample delivery unit orthe pump volume V thereof. The second connecting part, which consists ofan aspirating part 44 and a supply part 52, is implemented as separable.For this purpose, the supply part 52 opens into an injection port 45,which is connected via the supply part 52 to a second sample loop port13 of the injection valve 3. The aspirating part 44, which is connectedat one end to the pump volume V of the metering device 5, has a sampleneedle 42 at the other end, using which the aspirating part 44 can beconnected to the injection port 45.

However, the sample needle 42 can also be moved toward a samplecontainer 43 and can aspirate a defined sample volume therefrom—in themanner described hereafter—into the aspirating part 44. Furthermore, thesample needle 42 can also be moved toward a container for a flushingfluid (not shown), to take flushing liquid therefrom for a flushingoperation, using which the sample loops 51, 52, 44, the pump volume V,and optionally also the ports and grooves or channels of the injectionvalve can be cleaned. However, due to the special topology of theillustrated split loop principle, flushing of the sample loops 51, 52,44 and of the sample delivery unit 5 is normally not necessary, sincethe latter are flushed through with solvent, which is delivered by thepump 40, in any case during an injection operation. However, the outerside of the sample needle 42 can be cleaned by the immersion in acontainer having cleaning or flushing liquid. Alternatively, the needle42 can also be moved toward a washing and/or waste port (not shown inthe drawing), to be cleaned and/or to discard excess solvent.

The metering device 5 comprises a syringe 50 in the illustratedembodiment, in which a plunger 53 is guided in a pressure-tight anddisplaceable manner. The plunger 53 is driven by means of a drive 55,for example, a stepping motor. The drive 55 is activated by a controlunit 60. The control unit 60 also controls the switching operations ofthe injection valve 3 which has an activatable drive (not shown).

A waste port 12 of the injection valve is connected to a waste line 47,from which fluid can be discharged into a waste reservoir (not shown).

The high-pressure pump 40 is connected to a high-pressure port 15 of theinjection valve. A chromatography column 41 is connectable to thefurther high-pressure port 14. The high-pressure pump 40 can beintegrated as a component into the automatic sampler, however, it canalso be provided in another unit or a separate pump unit.

The injection valve 3 consists of a stator 1 and a rotor 2. The stator 1has the two high-pressure ports 14, 15, the two sample loop ports 13,16, and the waste port 12. Of course, instead of the illustratedinjection valve, injection valves having more than 5 ports are alsoconceivable for implementing the invention. Via these ports, theinjection valve 3 is connected via the above-described connecting lines,which can be implemented as capillary connections, to the otherfunctional elements of the HPLC system. The high-pressure screwconnections required for this purpose are not shown for the sake ofcomprehensibility in FIG. 1. For reasons of simplicity, the injectionvalve is shown in the interface between stator 1 and rotor 2, whereinboth the embodiment of the end face of the stator 1 and also theembodiment of the end face of the rotor 2 are shown to make it easier tounderstand the mode of operation. Inside the injection valve 3, theports are implemented as boreholes, which lead to the other side of thestator 1. The rotor 2 has a number of curved grooves 21, 23, 25, whichare aligned precisely with the boreholes of the input and output ports.

The rotor 2 is pressed with a contact pressure force against the stator1, so that a mutual interface between rotor 2 and stator 1 isimplemented, at which the two parts form a seal against one another. Thecontact pressure force is dimensioned in this case so that thearrangement is still leak-tight even at the highest pressures to beexpected.

In the so-called equilibration phase of the system illustrated in FIG.2, the switching or injection valve is located in the INJECT position,so that the pump actively flushes solvent through the sample loop in thedirection of the column. A required GDV of the entire system or of theautomatic sampler 10, which was determined during the method developmentor is already predefined, is adjusted by means of positioning of theplunger 53 during the equilibration (GDV of the entire system andadditional ΔGDV of the metering device 5).

In the illustrated exemplary embodiment, a position Pos. B is assumedfor this purpose, in which the metering device has a pump volume V_(B).This volume already contains a desired change ΔGDV (=V_(A)) of the GDVof the automatic sampler 10 (or of its conventional GDV) and a desiredsample quantity V_(sample) (=V_(B)−V_(A)) to be received (at a laterpoint in time), wherein, of course, other variants without including asample volume at such a point in time are also conceivable.

A varied piston positioning during the equilibration and a thereforevaried pump volume V of the volume adjustment device results in achanged pressure and flow (option 1), wherein the volume change can becompensated for in a preferred embodiment of the invention by adaptingthe flow rate, so that the pressure and flow still remain stable (option2). The adaptation of the flow rate is performed in this case byactivation of the pump 40 by the control unit 60, which opposes themetering device 5 accordingly.

Subsequently, the valve 3, as shown in FIG. 3, switches into a PRESSUREEQUALIZATION position, in which the connecting part 51 and the secondconnecting part or the supply part 52 of the sample loop have noconnection to the other components connected to the injection valve 3and the sample loop is therefore switched out of the analytical path.The sample loop also has system pressure at this time (high pressuregreater than 500 bar or even greater than 1500 bar).

In one variant (only required in the case of a method 1 explainedhereafter), the drive 55 of the metering device 5 can move forwardbriefly, until the plunger 53 moves. In this case, the force on theplunger 53 or the torque of the drive 55 is measured and stored. It is ameasure of the pressure in the sample loop (system pressure). In anothervariant, the pressure is ascertained or monitored by means of a sensoror the plunger position is detected and stored for later use.

Thereafter, the metering device 5 decompresses the sample loop bychanging the plunger position from position B to position C orincreasing the pump volume from V_(B) to V_(C) until almost toatmospheric pressure. In this case, the required movement path of theplunger 53 can be ascertained by measuring the force on the plunger 53or the drive torque during the aspiration of the preceding sample ordetermined by a pressure sensor in the sample loop.

As shown in FIG. 4, the switching valve 3 subsequently switches to theWASTE position, in which the quantity of solvent is expelled by themetering device 5, which corresponds to the sample quantity V_(sample)(=V_(C)−V_(A)) to be aspirated.

In the state shown in FIG. 5, the sample needle 42 was subsequentlymoved into the sample container 43, so that a sample volume can beaspirated. For this purpose, the plunger 53 is initially in position Aand is controlled by the control unit 60 for the aspiration in positionC. In this case, the desired, defined sample volume V_(sample)(=V_(C)−V_(A)) is aspirated into the aspirating part 44, wherein thevolume of the sample is preferably less than the volume of theaspirating part 44, so that no mixing of the sample fluid with the fluidconveyed by the high-pressure pump can occur in the pump volume. FIG. 5shows the state of the HPLC system after ending the aspirationoperation.

In the first LOAD position of the valve 3 shown in FIG. 5, the grooves21, 23, 25 are aligned with the ports 12 to 16 so that the grooves 23and 25 connect the two high-pressure ports 14, 15 or the waste port 12and the sample loop port 13. In this LOAD position, the high-pressurepump 40 therefore delivers fluid in the direction toward thechromatography column 41. Furthermore, the sample loop port 16 is closedpressure-tight.

During the aspirating, the force on the plunger 53 or the drive torqueof the metering device 5 can be measured, wherein the drive torque orthe plunger force represents a measure of the atmospheric pressure hereand can be used as explained above during the decompression. Of course,it is also conceivable to ascertain, monitor, and/or store for later usethe (atmospheric) pressure by means of a pressure sensor.

Subsequently, the switching valve 3 switches to a PRESSURE EQUALIZATIONposition, in which the connecting part 51 and the second connecting partor the supply part 52 of the sample loop do not have a connection to theother components connected to the injection valve 3. So as not tointerrupt the flow through the chromatography column 41 during thedelivery of the volume required for the compression of the sample loopcontent, the groove 25 in the rotor 2 of the valve is embodied aslengthened accordingly, so that the two high-pressure ports 14, 15 arealso still connected in the PRESSURE EQUALIZATION position.

The sample loop is compressed in this case in one variant (method 1)until the stored (as explained above) drive torque or the plunger forceis achieved as a measure of the system pressure. The pressure then againcorresponds to the system pressure (high pressure). A compressibility ofthe sample which deviates from the solvent can cause the resultingplunger position to no longer correspond precisely under certaincircumstances to the starting position, position B, of the plunger 53 inthis case. In this way, exact precompression of the sample loop to thesystem pressure is performed with a small negligible deviation of thedesired value GDV_(target) of the GDV.

In another conceivable variant (method 2), the plunger 53, as shown inFIG. 6, is navigated to the starting position, position B, again andthus compresses the sample loop. In this case, deviations of thecompressibility of the sample in comparison to the compressibility ofthe solvent result in slight deviations, which are to be neglected, fromthe system pressure. For this purpose, the achieved GDV correspondsexactly to the desired value GDV_(target) of the GDV.

To be able to inject the sample volume which is located in theaspirating part 44, the sample needle 42 is moved into the injectionport 45. This port seals off the needle tip so it is resistant to highpressure. Subsequently, the switching valve or injection valve 3switches to the inject position, in which the aspirated sample volume isconveyed completely out of the aspirating part 44 to the column 41(injection) by the solvent conveyed by the pump 40. If a gradient isnavigated for the solvent (chronologically controlled mixing ratio ofthe solvent) for a chromatography run, no undesired delaysadvantageously result.

In a preferred embodiment of the invention, a deviation of the GDVcaused by the precompression can be readjusted after the injection,wherein the correction movement of the plunger (to position B) iscompensated for by corresponding adaptation of the flow rate. In thisway, with exact precompression, an exact (adjusted or desired)GDV_(target) can nonetheless be obtained. In addition, it is alsopossible (in particular if the sample volume after the compression isgreater than the desired GDV_(target)) to readjust the desiredGDV_(target) during the chromatography run.

Although it is described differently in the exemplary embodiment,however, it is, of course, also conceivable to set or vary the GDV(deviating from a system-specific GDV_(actual)) to a desired amountGDV_(target) also during a chromatography run instead of before. Forthis purpose, the pump 40 can be activated in opposition to the changeof the metering device 5 accordingly via the control unit 60, to keepthe flow rate or the volume stream to the column 41 constant.

Optionally, at the end of the chromatography run, the deviation ΔGDV(=GDV_(target)−GDV_(actual)) and therefore GDV or the mixing volume canbe set to a minimum (minimal pump volume) by setting the plunger 53 atposition 0, to shorten the washing-out time of the system, in particularof the sample loop.

List of reference signs  1 stator  2 rotor  3 injection valve  5metering device 10 automatic sampler or chromatography system 12 wasteport 13 second sample loop port 14 further high-pressure port 15high-pressure port 16 first sample loop port 21, 23, 25 grooves 40high-pressure pump 41 separating column 42 sample needle 43 samplecontainer 44 aspirating part 45 injection port 47 waste line 50 syringe51 first connecting part 52 supply part 53 plunger 55 drive 60 controlunit V pump volume VA pump volume in position A VB pump volume inposition B Vc pump volume in end position C V_(sample) sample quantityV_(max) maximum volume of the metering device Pos. 0 lowermost positionof the plunger Pos. A position A of the plunger Pos. B position B of theplunger Pos. C end position of the plunger GDV gradient delay volume ofthe entire system GDV_(actual) system-specific value of the GDVGDV_(target) desired value of the GDV ΔGDV deviation of the GDV due tocorresponding plunger position

What is claimed is:
 1. A method for setting a gradient delay volume of aliquid chromatography system for a chromatography run, the methodcomprising: setting, and inputting into a control unit of the liquidchromatography system, a target gradient delay volume (GDV_(target)),wherein the target gradient delay volume (GDV_(target)) is predefinedbased on an earlier chromatography run of the liquid chromatographysystem; calculating, by the control unit, a difference of the gradientdelay volume (ΔGDV) between the target gradient delay volume(GDV_(target)) and an actual gradient delay volume (GDV_(actual)) of theliquid chromatography system for the chromatography run; controlling, bythe control unit, a metering device of the liquid chromatography system,the metering device includes a syringe and a plunger, the plunger isconfigured to be guided in a pressure-tight and displaceable manner,wherein controlling the metering device is accomplished by moving theplunger within the syringe, the metering device further including avariable pump volume V defined by the plunger, to adjust the pump volumeV by adjusting a position of the plunger within the syringe, in whichthe adjusted volume is equal to the calculated difference of thegradient delay volume; and performing the chromatography run on theliquid chromatography system using the adjusted volume and a solventgradient, the liquid chromatography system further comprising aninjection valve, a pump connected to the injection valve, a separationcolumn connected to the injection valve, and a sample loop extendingbetween a pair of sample loop ports of the injection valve, wherein thesample loop comprises the pump volume V of the metering device.
 2. Themethod of claim 1, in which the difference of the gradient delay volumeis calculated with an equation, the equation comprising:ΔGDV=GDV_(target)−GDV_(actual), where ΔGDV is the difference of thegradient delay volume due to the position of the plunger within themetering device, GDV_(target) is the target gradient delay volume, andGDV_(actual) is the gradient delay volume of the liquid chromatographysystem.
 3. The method of claim 1, in which the liquid chromatographysystem includes an injection port selectively connected to theseparation column via fluidly interconnected components, in which theactual gradient delay volume comprises a holding capacity of the fluidlyinterconnected components extending from the metering device to theseparation column.
 4. The method of claim 1, in which the difference ofthe gradient delay volume is less than or equal to 1000 microliters. 5.The method of claim 1, in which the performing of the chromatography runstep includes injecting the sample into the separation column, in whichthe setting of the target gradient delay volume occurs after the sampleinjection.
 6. The method of claim 5, in which the controlling themetering device step includes adjusting the plunger of the pump tocompensate for the adjusted volume V of the metering device so that aflow rate and a pressure of the sample entering the separation column isessentially constant.
 7. The method of claim 1, in which the performingof the chromatography run step includes injecting the sample into theseparation column, in which the setting of the target gradient delayvolume occurs before the sample injection.
 8. The method of claim 1,wherein the performing of the chromatography run step includes, at theend of the chromatography run, adjusting the position of the plunger inthe metering device so that the volume of the metering device is zero.9. The method of claim 1, in which the performing of the chromatographyrun step includes taking a sample into the sample loop with the meteringdevice.
 10. The method of claim 1, in which the sample loop furthercomprises: a first connecting part and a second connecting part, inwhich the first connecting part is connected to a first sample loop portof the pair of sample loop ports and to the metering device, in whichthe second connecting part is connected to a second sample loop port ofthe pair of sample loop ports and to the metering device, in which thesecond connecting part comprises an aspirating part and a supply part,in which the aspirating part and the supply part are configured to beseparated, in which the aspirating part is connected to the meteringdevice and the supply part is connected to the injection valve.